Luria-Bertani Agar Research Articles

Pseudomonas nitritireducens sp. nov., a nitrite reduction bacterium isolated from wheat soil

A Gram-negative, non-mobile, polar single flagellum, rod-shaped bacterium WZBFD3-5A2(T) was isolated from a wheat soil subjected to herbicides for several years. Cells of strain WZBFD3-5A2(T) grow optimally on Luria-Bertani agar medium at 30 °C in the presence of 0-4.0 % (w/v) NaCl and pH 8.0. 16S rRNA gene sequence analysis revealed that strain WZBFD3-5A2(T) belongs to the genus Pseudomonas. Physiological and biochemical tests supported the phylogenetic affiliation. Strain WZBFD3-5A2(T) is closely related to Pseudomonas nitroreducens IAM1439(T), sharing 99.7 % sequence similarity. DNA-DNA hybridization experiments between the two strains showed only moderate reassociation similarity (33.92 ± 1.0 %). The DNA G+C content is 62.0 mol%. The predominant respiratory quinine is Q-9. The major cellular fatty acids present are C(16:0) (28.55 %), C(16:1ω6c) or C(16:1ω7c) (20.94 %), C(18:1ω7c) (17.21 %) and C(18:0) (13.73 %). The isolate is distinguishable from other related members of the genus Pseudomonas on the basis of phenotypic and biochemical characteristics. From the genotypic, chemotaxonomic and phenotypic data, it is evident that strain WZBFD3-5A2(T) represents a novel species of the genus Pseudomonas, for which the name Pseudomonas nitritereducens sp. nov. is proposed. The type strain is WZBFD3-5A2(T) (=CGMCC 1.10702(T) = LMG 25966(T)).

First Report of Bacterial Black Spot Disease in Watermelon Caused by Acidovorax valerianellae in Korea.

Watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai), an important member of the Cucurbitaceae family, is cultivated on 21,000 ha that produces 850,000 t in Korea. In April 2011, we received grafted watermelon with necrotic leaf spots from a commercial watermelon grower in Andong, Korea. Black spots were observed on cotyledons of the plants in seedbeds, and approximately 9% of watermelon plants were infected with the disease. Initial symptoms on the seedling were black, greasy spots sometimes surrounded by a halo of discoloration. Younger leaves usually showed symptoms later than cotyledons. Bacteria isolated from the infected plants were gram-negative, motile, straight rods with a single flagellum and 0.84 to 0.89 μm wide and 1.54 to 1.69 μm long. They formed rough colonies with a white-cream color after 48 h of incubation on Luria-Bertani (LB) agar at 28°C. Colonies of isolates were nonfluorescent, smooth, and white on King's medium B. On YBGA (7 g of yeast extract, 7 g of bactopeptone, 7 g of glucose, 15 g of agar, 1,000 ml of distilled water; pH 7.2) colonies are circular, raised with an entire margin, and white to cream. Pathogenicity tests were conducted with potted, greenhouse-grown watermelon plants. Bacterial colonies grown on LB medium for 48 h at 28°C were suspended in sterile distilled water, and the suspension (1.0 × 108 CFU/ml) was infiltrated into mesophyll of watermelon leaves with a syringe as previously described (2). Inoculated plants were maintained at 28°C and 90% relative humidity in a growth chamber with a daily 12-h photoperiod of fluorescent light. Five plants were used for inoculation. Sterilized distilled water was used as a control. The bacterial isolates induced necrosis in the infiltrated area within 3 to 5 days. Typical water-soaked spots appeared after 3 days of incubation and became gray to black after 6 days. The bacterium was successfully reisolated from the diseased lesions, thus completing Koch's postulates. A cell suspension (50 μl of 1 × 106 CFU/ml) was infiltrated with a syringe into the intercellular spaces of tobacco leaves to determine the hypersensitive reaction (HR). A typical HR developed 20 h after leaf infiltration. The 16S rDNA region of the isolates, amplified by using universal PCR primers, shared 99% sequence identity with an Acidovorax valerianellae strain (GenBank Accession No. AJ431731) (1). The resulting sequences of 1,424 bp were deposited in GenBank (Accession No. JN983471). The isolates we obtained in this study clustered with A. valerianellae on a phylogenetic tree generated by the neighbor-joining method implemented in MEGA Version 4.1. In the Biolog Microbial Identification System, Version 4.2 (Biolog Inc., Hayward, CA), all isolates were 63 to 77% similar with a match probability of 100% to A. konjaci. Fatty acid composition analysis of isolates based on the MIDI Library version TSBA 5.0 and Library Generation system software version 5.0 showed that the isolates were 52 and 72% similar to an Acidovorax sp., respectively. To our knowledge, this is the first report of bacterial black spot disease in watermelon caused by A. valerianellae in Korea. A. valerianellae is a causal agent of bacterial black spot in corn salad and is transmitted by inoculated seeds (3). Further studies are required to determine whether it is seed transmitted in watermelon.

Midgut bacterial dynamics in Aedes aegypti

In vector mosquitoes, the presence of midgut bacteria may affect the ability to transmit pathogens. We have used a laboratory colony of Aedes aegypti as a model for bacterial interspecies competition and show that after a blood meal, the number of species (culturable on Luria-Bertani agar) that coexist in the midgut is low and that about 40% of the females do not harbor any cultivable bacteria. We isolated species belonging to the genera Bacillus, Elizabethkingia, Enterococcus, Klebsiella, Pantoea, Serratia, and Sphingomonas, and we also determined their growth rates, antibiotic resistance, and ex vivo inhibition of each other. To investigate the possible existence of coadaptation between midgut bacteria and their host, we fed Ae.aegypti cohorts with gut bacteria from human, a frog, and two mosquito species and followed the bacterial population growth over time. The dynamics of the different species suggests coadaptation between host and bacteria, and interestingly, we found that Pantoea stewartii isolated from Ae.aegypti survive better in Ae.aegypti as compared to P.stewartii isolated from the malaria mosquito Anopheles gambiae.

Comparative study of the antibacterial penetrating effects of wound dressings

In an infected wound, bacteria are present not only on the surface but also within the granulation tissue. The presence of bacteria inside the granulation tissue is one of the reasons for our inability to control infection. We have developed an in vitro model of an infected wound using Luria-Bertani (LB) agar media and non-pathogenic Escherichia coli, and compared the differences in the antibacterial effects of various types of antibacterial wound dressings. The results have shown that the penetrating antibacterial effects differed according to the type of wound dressing selected. As the thickness of the layer of the LB agar media increased, the potency of antibacterial effects caused by penetration decreased. In conclusion, it was suggested that antibacterial wound dressings can have antibacterial effects against bacteria not only on the surface of an infected wound but inside, so it is necessary to use different wound dressings according to the conditions of each infected wound.

Isolation and characterization of a novel α-amylase from a metagenomic library of Western Ghats of Kerala, India

In the present study, metagenomic library of Western Ghats soil sample was constructed in a fosmid vector (pCC1FOS) and screened for biocatalytic properties. The clones showed amylolytic activity on Luria-Bertani starch agar plates and one of them was studied in detail. The enzyme exhibited stability at elevated temperature with 60°C being the optimal temperature. The enzyme retained more than 30% activity after 60 min incubation at 80°C. It also showed more than 70% activity retention in 1.5 M NaCl solution. The pH optimum of the enzyme was at pH = 5.0. The enzyme possesses good activity in the presence of chelating and strong reducing agents with activity enhancements or retention being observed at 5 mM β-mercaptoethanol, dithiothreitol and N-bromosuccinimide. However, almost complete loss of activity was observed with 5 mM EDTA, while activity enhancement was observed upon incubation with Ca2+ suggesting it to be a Ca2+-dependent α-amylase, which was further confirmed by a thin-layer chromatography (TLC). The TLC run revealed that digestion pattern was similar to commercial α-amylase. The 16S rRNA gene sequence (GenBank accession number HQ680979) BLAST showed 95% similarities with Exiguobacterium sp. AFB-11 and AFB 18, with query sequence coverage of 99%.

Discrimination of bacteria from Jamaican bauxite soils using laser-induced breakdown spectroscopy

Soil bacteria are sensitive to ecological change and can be assessed to gauge anthropogenic influences and ecosystem health. In recent years, there has been a significant increase in the focus on new technologies that can be applied to the evaluation of soil quality. Laser-induced breakdown spectroscopy (LIBS) is a promising technique that has been used for the investigation and characterization of explosives, solids, liquids, gases, biological and environmental samples. In this study, bacteria from un-mined and a chronosequence of reclaimed bauxite soils were isolated on Luria-Bertani agar media. Polymerase chain reaction amplification of the bacterial 16S rDNA, sequencing, and phylogenetic analysis were applied to each isolated soil bacteria from the sample sites resulting in the identification and classification of the organisms. Femtosecond LIBS performed on the isolated bacteria showed atomic and ionic emission lines in the spectrum containing inorganic elements such as sodium (Na), magnesium (Mg), potassium (K), zinc (Zn), and calcium (Ca). Principal component analysis and partial least squares regression analysis were performed on the acquired bacterial spectra demonstrating that LIBS has the potential to differentiate and discriminate among bacteria in the un-mined and reclaimed chronosequence of bauxite soils.

A novel subtilase with NaCl-activated and oxidant-stable activity from Virgibacillussp. SK37

BackgroundMicrobial proteases are one of the most commercially valuable enzymes, of which the largest market share has been taken by subtilases or alkaline proteases of the Bacillus species. Despite a large amount of information on microbial proteases, a search for novel proteases with unique properties is still of interest for both basic and applied aspects of this highly complex class of enzymes. Oxidant stable proteases (OSPs) have been shown to have a wide application in the detergent and bleaching industries and recently have become one of the most attractive enzymes in various biotechnological applications.ResultsA gene encoding a novel member of the subtilase superfamily was isolated from Virgibacillus sp. SK37, a protease-producing bacterium isolated from Thai fish sauce fermentation. The gene was cloned by an activity-based screening of a genomic DNA expression library on Luria-Bertani (LB) agar plates containing 1 mM IPTG and 3% skim milk. Of the 100,000 clones screened, all six isolated positive clones comprised one overlapping open reading frame of 45% identity to the aprX gene from Bacillus species. This gene, designated aprX-sk37 was cloned into pET21d(+) and over-expressed in E. coli BL21(DE3). The enzyme product, designated AprX-SK37, was purified by an immobilized metal ion affinity chromatography to apparent homogeneity and characterized. The AprX-SK37 enzyme showed optimal catalytic conditions at pH 9.5 and 55°C, based on the azocasein assay containing 5 mM CaCl2. Maximum catalytic activity was found at 1 M NaCl with residual activity of 30% at 3 M NaCl. Thermal stability of the enzyme was also enhanced by 1 M NaCl. The enzyme was absolutely calcium-dependent, with optimal concentration of CaCl2 at 15 mM. Inhibitory effects by phenylmethanesulfonyl fluoride and ethylenediaminetetraacetic acid indicated that this enzyme is a metal-dependent serine protease. The enzyme activity was sensitive towards reducing agents, urea, and SDS, but relatively stable up to 5% of H2O2. Phylogenetic analysis suggested that AprX-SK37 belongs to a novel family of the subtilase superfamily. We propose the name of this new family as alkaline serine protease-X (AprX).ConclusionsThe stability towards H2O2 and moderately halo- and thermo-tolerant properties of the AprX-SK37 enzyme are attractive for various biotechnological applications.

Biotinylation, a Post-translational Modification Controlled by the Rate of Protein-Protein Association

Biotin protein ligases catalyze specific covalent linkage of the coenzyme biotin to biotin-dependent carboxylases. The reaction proceeds in two steps, including synthesis of an adenylated intermediate followed by biotin transfer to the carboxylase substrate. In this work specificity in the transfer reaction was investigated using single turnover stopped-flow and quench-flow assays. Cognate and noncognate reactions were measured using the enzymes and minimal biotin acceptor substrates from Escherichia coli, Pyrococcus horikoshii, and Homo sapiens. The kinetic analysis demonstrates that for all enzyme-substrate pairs the bimolecular rate of association of enzyme with substrate limits post-translational biotinylation. In addition, in noncognate reactions the three enzymes displayed a range of selectivities. These results highlight the importance of protein-protein binding kinetics for specific biotin addition to carboxylases and provide one mechanism for determining biotin distribution in metabolism.

Development of a high-throughput screening method for recombinant Escherichia coli with intracellular dextransucrase activity

To efficiently engineer intracellular dextransucrase (DSase) expression in Escherichia coli, a high-throughput screening method was developed based on the polymer-forming activity of the enzyme. Recombinant E. coli containing the Leuconostoc citreum DSase (LcDS) gene was grown on Luria-Bertani agar plates, containing 2% sucrose, at 37°C for 8 h. The plates were then evenly overlaid with 0.6% soft agar, containing 1.2 mg/ml D-cycloserine, and incubated at 30°C to allow gradual cell disruption until a dextran polymer grew through the overlaid layer. A significant correlation between dextran size and enzyme activity was established and applied for screening truncated mutants with LcDS activity.

Study of the Thiol/Disulfide Redox Systems of the Anaerobe Desulfovibrio vulgaris Points Out Pyruvate:Ferredoxin Oxidoreductase as a New Target for Thioredoxin 1

Sulfate reducers have developed a multifaceted adaptative strategy to survive against oxidative stresses. Along with this oxidative stress response, we recently characterized an elegant reversible disulfide bond-dependent protective mechanism in the pyruvate:ferredoxin oxidoreductase (PFOR) of various Desulfovibrio species. Here, we searched for thiol redox systems involved in this mechanism. Using thiol fluorescent labeling, we show that glutathione is not the major thiol/disulfide balance-controlling compound in four different Desulfovibrio species and that no other plentiful low molecular weight thiol can be detected. Enzymatic analyses of two thioredoxins (Trxs) and three thioredoxin reductases allow us to propose the existence of two independent Trx systems in Desulfovibrio vulgaris Hildenborough (DvH). The TR1/Trx1 system corresponds to the typical bacterial Trx system. We measured a TR1 apparent K(m) value for Trx1 of 8.9 μM. Moreover, our results showed that activity of TR1 was NADPH-dependent. The second system named TR3/Trx3 corresponds to an unconventional Trx system as TR3 used preferentially NADH (K(m) for NADPH, 743 μM; K(m) for NADH, 5.6 μM), and Trx3 was unable to reduce insulin. The K(m) value of TR3 for Trx3 was 1.12 μM. In vitro experiments demonstrated that the TR1/Trx1 system was the only one able to reactivate the oxygen-protected form of Desulfovibrio africanus PFOR. Moreover, ex vivo pulldown assays using the mutant Trx1(C33S) as bait allowed us to capture PFOR from the DvH extract. Altogether, these data demonstrate that PFOR is a new target for Trx1, which is probably involved in the protective switch mechanism of the enzyme.

Microbial toxicity of metal oxide nanoparticles (CuO, NiO, ZnO, and Sb2O3) to Escherichia coli, Bacillus subtilis, and Streptococcus aureus

In this study, the microbial toxicities of metal oxide nanoparticles were evaluated for Escherichia coli, Bacillus subtilis, and Streptococcus aureus in laboratory experiments. The nanoparticles tested were CuO, NiO, ZnO, and Sb2O3. The metal oxide nanoparticles were dispersed thoroughly in a culture medium, and the microorganisms were cultivated on Luria–Bertani agar plates containing different concentrations of metal oxide nanoparticles. The bacteria were counted in terms of colony forming units (CFU). The CFU was reduced in a culture medium containing metal oxide NP, and the dose–response relationship was characterized. CuO nanoparticles were found to be the most toxic among the tested nanoparticles, followed by ZnO (except S. aureus), NiO, and Sb2O3 nanoparticles. We determined that the intrinsic toxic properties of heavy metals are also associated with the toxicity of metal oxide nanoparticles. Ion toxicity was also evaluated to determine the effects of metal ions dissolved from metal oxide NPs, and the toxicity induced from the dissolved ions was determined to be negligible herein. To the best of our knowledge, this is the first study of the toxicity of NiO and Sb2O3 NPs on microorganisms. We also discuss the implications of our findings regarding the effects of the intrinsic toxic properties of heavy metals, and concluded that the apparent toxicities of metal oxide NPs can largely be understood as a matter of particle toxicity.

Novel listerial genetic loci conferring enhanced barotolerance in Escherichia coli

To identify Listeria monocytogenes genes with a role in high-pressure (HP) resistance. A L. monocytogenes genomic library constructed in an Escherichia coli background was screened for loci conferring increased HP resistance. Pressure treatments at 400 megapascals for 5 min in Luria-Bertani (LB) agar were used to screen for increased resistance to pressure. Colonies arising on the treated agar plates were isolated, the plasmid extracted and the inserts sequenced to identify the genetic loci conferring HP resistance. Seven different genetic regions were identified, which encoded proteins similar to an inorganic polyphosphate/ATP-NAD kinase, the septation ring formation regulator EzrA, flagellar motor proteins MotA and MotB, proteins similar to the quorum sensing Agr system from Staphylococcus (AgrA, AgrC and AgrD), proteins similar to a transcription regulator (RpiR family) and a fructose phosphotransferase system, proteins of unknown function, and a Fur regulator. Of the seven loci confirmed, three (EzrA, MotA/B and the Agr system) maintained significantly reproducible HP tolerance when expressed in a different E. coli background. Novel genetic loci from the L. monocytogenes genome confer increased HP resistance when heterologously expressed in an E. coli background. Molecular and functional approaches to the screening of genetic elements linked to HP resistance provide greater insights into microbial inactivation and/or survival mechanisms when using HP as a means of controlling/eliminating bacterial growth. This information will ultimately have significant implications for the use of HP processing in the food industry, in terms of both food quality and safety.

The Response of Shewanella oneidensis MR-1 to Cr(III) Toxicity Differs from that to Cr(VI)

Chromium is a contaminant of concern that is found in drinking water in its soluble, hexavalent form [Cr(VI)] and that is known to be toxic to eukaryotes and prokaryotes. Trivalent chromium [Cr(III)] is thought to be largely harmless due to its low solubility and inability to enter cells. Previous work has suggested that Cr(III) may also be toxic to microorganisms but the mechanism remained elusive. In this work, we probe the toxicity of Cr(III) to Shewanella oneidensis MR-1, a bacterium able to reduce Cr(VI) to Cr(III) and compare it to Cr(VI) toxicity. We found evidence for Cr(III) toxicity both under Cr(VI) reducing conditions, during which Cr(III) was generated by the reduction process, and under non-reducing conditions, when Cr(III) was amended exogenously. Interestingly, cells exposed to Cr(III) (200 μM) experienced rapid viability loss as measured by colony forming units on Luria–Bertani (LB) agar plates. In contrast, they maintained some enzymatic activity and cellular integrity. Cr(VI)-exposed cells exhibited loss of enzymatic activity and cell lysis. The loss of viability of Cr(III)-exposed cells was not due to membrane damage or to enzymatic inhibition but rather appeared to be associated with an abnormal morphology that consisted of chains of membrane-enclosed units of irregular size. Exposure of abnormal cells to growth conditions resulted in membrane damage and cell death, which is consistent with the observed viability loss on LB plates. While Cr(VI) was taken up intracellularly and caused cell lysis, the toxic effect of Cr(III) appeared to be associated with extracellular interactions leading to an ultimately lethal cell morphology.

The genetic environment of armA on pHNE, an IncN plasmid, in one Escherichia coli isolate from a chicken

Sir, Methylation of 16S ribosomal RNA (rRNA) mediated by 16S rRNA methylase, which confers high-level aminoglycoside resistance (MICs .1024 mg/L) in Enterobacteriaceae, has recently emerged as a major medical problem worldwide. Until now, seven plasmid-encoded 16S rRNA methylases, ArmA, RmtA, RmtB, RmtC, RmtD, RmtE and NpmA, have been reported in various clinical Gram-negative isolates in multiple geographic regions. armA is one of the most prevalent 16S rRNA methylase genes found in several Gram-negative pathogens. Since it was initially identified in Klebsiella pneumoniae in 2003, two studies focusing on its genetic environment and dissemination in Enterobacteriaceae from both humans and animals have been conducted. Here, we identify a novel genetic environment of armA on pHNE, an IncN plasmid, in one Escherichia coli isolate from a chicken. Previously, we reported the emergence of 16S rRNA methylases encoded by armA and rmtB in E. coli isolates from chickens. In a routine survey of antimicrobial-resistant isolates from chickens in 2009, armA was detected in an E. coli isolate exhibiting high-level resistance to the aminoglycoside antibiotic amikacin (MICs .1024 mg/L) (n1⁄41/18) from a diseased flock in a typical traditional farm in Henan Province, China. Phylogenetic group analysis using multiplex PCR revealed that this armA E. coli isolate belonged to phylogenetic group D, which was associated with extra-intestinal virulence. In order to further elucidate the genetic environment of armA, plasmids extracted from this armA-positive isolate (using the Plasmid Midi Kit, Qiagen, Germany) were transferred to E. coli DH10B by electroporation to investigate whether the armA resistance determinant was localized on plasmids. The transformants were screened on a Luria–Bertani agar plate containing

Streptococcus pyogenes Ser/Thr Kinase-regulated Cell Wall Hydrolase Is a Cell Division Plane-recognizing and Chain-forming Virulence Factor

Cell division and cell wall synthesis are closely linked complex phenomena and play a crucial role in the maintenance and regulation of bacterial virulence. Eukaryotic-type Ser/Thr kinases reported in prokaryotes, including that in group A Streptococcus (GAS) (Streptococcus pyogenes Ser/Thr kinase (SP-STK)), regulate cell division, growth, and virulence. The mechanism of this regulation is, however, unknown. In this study, we demonstrated that SP-STK-controlled cell division is mediated under the positive regulation of secretory protein that possesses a cysteine and histidine-dependent aminohydrolases/peptidases (CHAP) domain with functionally active cell wall hydrolase activity (henceforth named as CdhA (CHAP-domain-containing and chain-forming cell wall hydrolase). Deletion of the CdhA-encoding gene resulted in severe cell division and growth defects in GAS mutants. The mutant expressing the truncated CdhA (devoid of the CHAP domain), although displayed no such defects, it became attenuated for virulence in mice and highly susceptible to cell wall-acting antibiotics, as observed for the mutant lacking CdhA. When CdhA was overexpressed in the wild-type GAS as well as in heterologous strains, Escherichia coli and Staphylococcus aureus, we observed a distinct increase in bacterial chain length. Our data reveal that CdhA is a multifunctional protein with a major function of the N-terminal region as a cell division plane-recognizing domain and that of the C-terminal CHAP domain as a virulence-regulating domain. CdhA is thus an important therapeutic target.

10% Povidone-Iodine May Be a Practical Field Water Disinfectant

A paucity of data exists regarding the use of iodophores such as povidone-iodine (PVI) to disinfect water. We sought to determine a practical minimal disinfecting concentration of 10% PVI over different contact times and temperatures when added to water inoculated with E. coli. 1:100, 1:1,000, and 1:10,000 dilutions of 10% PVI were created. Escherichia coli was exposed to these dilutions for 5, 15, and 30 minutes at 10, 20, and 30°C. Bactericidal activity was neutralized with 0.5% sodium thiosulfate. Mean viable colony forming units (CFUs) was determined after triplicate plating on Luria-bertani agar and 24 hours of incubation at 37°C. Effective bactericidal activity was defined as a 5-log reduction. Of the 200,000 E. coli plated, no CFUs were observed after exposure to the 1:100 dilution. After 5 minutes of contact time with the 1:1,000 dilution, at 10°C CFUs were too numerous to count (TNTC), at 20°C the mean CFU count was 92 (standard error ±11), and at 30°C the mean CFU count was 25 (standard error ±8). No CFUs were observed after 15 minutes of exposure to the 1:1,000 dilution across experimental temperatures. The 1:10,000 dilution always yielded CFU growth that was TNTC. The lowest disinfecting concentration of 10% PVI was the 1:1,000 dilution at 15 minutes of contact time. This supports the use of PVI for water disinfection against E. coli, the organism most commonly responsible for traveler's diarrhea. Further studies may assess its effectiveness against more virulent water borne pathogens.

The Protein-disulfide Isomerase DsbC Cooperates with SurA and DsbA in the Assembly of the Essential β-Barrel Protein LptD

The assembly of the β-barrel proteins present in the outer membrane (OM) of Gram-negative bacteria is poorly characterized. After translocation across the inner membrane, unfolded β-barrel proteins are escorted across the periplasm by chaperones that reside within this compartment. Two partially redundant chaperones, SurA and Skp, are considered to transport the bulk mass of β-barrel proteins. We found that the periplasmic disulfide isomerase DsbC cooperates with SurA and the thiol oxidase DsbA in the folding of the essential β-barrel protein LptD. LptD inserts lipopolysaccharides in the OM. It is also the only β-barrel protein with more than two cysteine residues. We found that surAdsbC mutants, but not skpdsbC mutants, exhibit a synthetic phenotype. They have a decreased OM integrity, which is due to the lack of the isomerase activity of DsbC. We also isolated DsbC in a mixed disulfide complex with LptD. As such, LptD is identified as the first substrate of DsbC that is localized in the OM. Thus, electrons flowing from the cytoplasmic thioredoxin system maintain the integrity of the OM by assisting the folding of one of the most important β-barrel proteins.

Transurethral Induction of Mouse Urinary Tract Infection

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on Luria-Bertani (LB) agar at 37 degrees C in ambient air. UPEC strains grow as yellowish-white translucent colonies on LB agar. Following confirmation of appropriate colony morphology, single colonies are then picked to be cultured in broth. LB broth can be used for most uropathogenic bacterial strains. Two serial, overnight LB broth cultures can be employed to enhance expression of type I pili, a well-defined virulence factor for uropathogenic bacteria. Broth cultures are diluted to the desired concentration in phosphate buffered saline (PBS). Eight to 12 week old female mice are placed under isoflurane anesthesia and transurethrally inoculated with bacteria using polyethylene tubing-covered 30 gauge syringes. Typical inocula, which must be empirically determined for each bacterial/mouse strain combination, are 10(6) to 10(8) cfu per mouse in 10 to 50 microliters of PBS. After the desired infection period (one day to several weeks), urine samples and the bladder and both kidneys are harvested. Each organ is minced, placed in PBS, and homogenized in a Blue Bullet homogenizer. Urine and tissue homogenates are serially diluted in PBS and cultured on appropriate agar. The following day, colony forming units are counted.

Bacterial Diversity and Distribution from the Whole Mite Extracts in Acaricide Resistant and Susceptible Populations of Twospotted Spider Mite-Tetranychus urticae (Acari: Tetranychidae)

Investigations of the diversity of bacteria in acaricide resistant and susceptible populations from the whole mite extracts of twospotted spider mite-Tetranychus urticae were made using 8 different bacterial growth media and identified through 16S rRNA gene sequence analysis. The pyridaben resistant population was diversified with six genera including Chryseobacterium, Sphingomonas, Acidovorax, Herbaspirillum, Janthinobacterium, and Xenophilus, whereas the acequinocyl resistant population was associated with Bacillus, Pasteurella, Staphylococcus, Enterobacter, and Pseudomonas. The fenpropathrin resistant population harbored only two phylotypes (Pantoea and Pseudomonas). Citrobacter, Enterobacter, and Pantoea were recovered from the susceptible population. No correlation was observed between bacterial density and bacterial diversity. Of the 8 different growth media, R2A supported a greater number of cultivable bacterial phylotypes followed by Luria Bertani and brain heart infusion agar. This study suggests that knowledge of the diversity of bacterial phylotypes present in host insect pest species may be useful for developing biological approaches in insect-microbe interaction.

Synthesis and applications of polyacrylamide gels catalyzed by silver nitrate

AbstractPolyacrylamide gels are widely used as matrices for biomolecular analysis and fractionation, and they are being developed as biomaterials for diverse medical and industrial applications. This study reports silver nitrate as a novel catalyst for the synthesis of polyacrylamide gels from acrylamide and N,N‐methylene bisacrylamide monomers. The conditions were defined for silver‐catalyzed, free‐radical‐induced polymerization, and a suitable buffer system was devised for the electrophoretic resolution of nucleic acids. A silver‐staining procedure was modified for these gels, and they were compared with N,N,N′,N′‐tetramethylethylenediamine‐catalyzed gels for sensitivity and gel background. Silver nitrate and ammonium persulfate at final concentrations of 100 and 625 μg/mL, respectively, polymerized the resolving gels within 20 min at room temperature. These gels exhibited antimicrobial properties. The gels with ≥10 μg/mL silver nitrate showed a zone of complete inhibition of Staphylococcus aureus growth on a Luria–Bertani agar plate. The silver‐catalyzed gels were also suitable as antigen‐ and drug‐delivery devices. Silver, acting as both a catalyst and a microbicidal agent, was better than N,N,N′,N′‐tetramethylethylenediamine for the synthesis of polyacrylamide gels as drug‐ and oxygen‐delivery devices for topical applications. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011